Media For Membrane Ion Exchange Chromatography Based On Polymeric Primary Amines, Sorption Device Containing That Media, And Chromatography Scheme And Purification Method Using The Same

ABSTRACT

Media and devices, such as anion exchangers including such media, wherein the media is a membrane having a surface coated with a polymer such as a polyallylamine. The resulting membrane offers stronger binding of protein impurities and superior removal of host cell proteins from biological samples than conventional ligands based on quaternary ammonium salts, including trimethylammonium ligands. Also described is a chromatography scheme and method for purifying monoclonal antibodies, wherein the anion exchange sorber is placed downstream of an affinity column (e.g., Protein A or Protein G) and optionally one or more polishing devices such as cationic exchange columns. Little or no dilution of the cation exchanger pool (or affinity column exchange pool where no cation exchanger is used) is necessary to lower the conductivity of the sample. The sorber functions well to strongly bind host cell proteins and other impurities in biological samples even at high conductivities and pH.

This application is a divisional of U.S. patent application Ser. No.13/167,297 filed Jun. 23, 2011, which is a divisional of U.S. patentapplication Ser. No. 12/221,496 filed Aug. 4, 2008, which claimspriority of U.S. Provisional application Ser. No. 60/964,653 filed Aug.14, 2007 and U.S. Provisional application Ser. No. 61/070,708 filed onMar. 25, 2008, the disclosures of which are incorporated herein byreference.

BACKGROUND OF THE INVENTION

This invention relates to anion exchange chromatography media based onpolymeric primary amines, an anion exchange sorber including that media,and a chromatography scheme including the sorber. Absorption refers totaking up of matter by permeation into the body of an absorptivematerial. Adsorption refers to movement of molecules from a bulk phaseonto the surface of an adsorptive media. Sorption is a general term thatincludes both adsorption and absorption. Similarly, a sorptive materialor sorption device herein denoted as a sorber, refers to a material ordevice that either ad- or absorbs or both ad- and absorbs. The media isparticularly applicable as a porous membrane sorber used in a flowthrough cartridge and more particularly to a cartridge free of aseparate exterior housing.

Strong anion exchangers, such as those based on quarternary ammoniumions, are used in downstream processing as a polishing media forcapturing negatively charged large impurities, such as endotoxins,viruses, nucleic acids, and host cell proteins (HCP) that are present influids such as biological fluids, particularly solutions of manufacturedbiotherapeutics Traditionally, anion exchangers have been offered andused in the bead format, for example Q Sepharose® available from GEHealthcare Bio-Sciences AB. However, throughput limitations ofbead-based systems require large volume columns to effectively captureimpurities.

In bead-based chromatography, most of the available surface area foradsorption is internal to the bead. Consequently, the separation processis inherently slow since the rate of mass transport is typicallycontrolled by pore diffusion. To minimize this diffusional resistanceand concomitantly maximize dynamic binding capacity, small diameterbeads can be employed. However, the use of small diameter beads comes atthe price of increased column pressure drop. Consequently, theoptimization of preparative chromatographic separations often involves acompromise between efficiency/dynamic capacity (small beads favored) andcolumn pressure drop (large beads favored).

In contrast, membrane-based chromatographic systems (also calledmembrane sorbers), have the ligands attached directly to the convectivemembrane pores, thereby eliminating the effects of internal porediffusion on mass transport. Additionally, the use of microporousmembrane substrates with a tight membrane pore size distribution coupledwith effective flow distributors can minimize axial dispersion andprovide uniform utilization of all active sites. Consequently, masstransfer rates of membrane sorber media may be an order of magnitudegreater than that of standard bead-based chromatography media, allowingfor both high efficiency and high-flux separations. Since single or evenstacked membranes are very thin compared to columns packed withbead-based media, reduced pressure drops are found along thechromatographic bed, thus allowing increased flow rates andproductivities. The necessary binding capacity is reached by usingmembranes of sufficient internal surface area, yielding deviceconfigurations of very large diameter to height ratios (d/h).

Properly designed membrane sorbers have chromatographic efficienciesthat are 10-100 times better than standard preparative bead-basedresins. Consequently, to achieve the same level of separation on amembrane sorber, a bed height 10-fold less can be utilized. Bed lengthsof 1-5 mm are standard for membrane sorbers, compared to bed heights of10-30 cm for bead-based systems. Due to the extreme column aspect ratiosrequired for large-volume membrane sorbers, device design is critical.To maintain the inherent performance advantages associated with membranesorbers, proper inlet and outlet distributors are required toefficiently and effectively utilize the available membrane volume.Membrane sorber technology is ideally suited for this application.Current commercial membrane sorbers, however, suffer from variousdrawbacks, including low binding strength, difficulty in removingviruses, endotoxins and nucleic acids.

A membrane sorber is a highly porous, interconnected media that has theability to remove (ad- and/or absorb) some components of a solution whenthe latter flows through its pores. The properties of the membranesorber and its ability to perform well in the required applicationdepend on the porous structure of the media (skeleton) as well as on thenature of the surface that is exposed to the solution. Typically, theporous media is formed first, from a polymer that does not dissolve orswell in water and possesses acceptable mechanical properties. Theporous media is preferably a porous membrane sheet made by phaseseparation methods well known in the art. See, for example, Zeman L J,Zydney A L, Microfiltration and Ultrafiltration: Principles andApplications, New York: Marcel Dekker, 1996. Hollow fiber and tubularmembranes are also acceptable skeletons. A separate processing step isusually required to modify the external or facial surfaces and theinternal pore surfaces of the formed porous structure to impart thenecessary adsorptive properties. Since the membrane structure is oftenformed from a hydrophobic polymer, another purpose of the surfacemodification step is also to make the surfaces hydrophilic, orwater-wettable.

There exist a number of approaches to modify the external or facialsurfaces and the internal pore surfaces of a membrane. Those skilled inthe art will readily recognize exemplary methods involving adsorption,plasma oxidation, in-situ free-radical polymerization, grafting andcoating. The majority of these methods lead to formation ofmonolayer-like structures on the membrane surface, which most of thetime achieve the goal of making it hydrophilic, yet fail to impartacceptable adsorptive properties, for example high capacity for theadsorbate. The capacity is defined as the amount (weight) of theadsorbate that can be retained by a given volume of the media. As longas all adsorption occurs on the membrane surface, the capacity will belimited by the membrane surface area. By their nature, microporousmembranes have lower surface area compared to chromatography beads. Oneway to increase surface area is to reduce pore size, which obviouslyleads to significant losses in flux. For example, the maximum(monolayer) adsorption of protein on a 0.65 um polyethylene membrane(Entegris Corp, Billerica Mass.) is about 20 mg/ml, regardless of thetype of surface interactions. This is significantly less than, forexample, agarose chromatography beads, with typical capacity about 80mg/ml.

The type of surface interactions driving the adsorption is defined bythe specific application in which a given membrane sorber product isused. Currently, there is a need for a high-capacity, high-affinitysorber that removes viruses, nucleic acids, endotoxins, and host cellproteins (HCPs) from a solution of monoclonal antibodies (MABs). Theseimpurities tend to have a lower isoelectric point than the MABs, whichmeans that at a certain pH they will be negatively charged while the MABwill be positively charged. An anion exchanger, i.e. a media that bearsa positive charge and attracts anions, is required to remove theseimpurities. A number of chemical moieties bear a positive charge in anaqueous solution, including primary, secondary, and tertiary amines, aswell as quaternary ammonium salts. The amines are positively charged atpH below 11, while the ammonium salts bear the positive charge at allpH, so these groups are commonly called weak and strong anionexchangers, respectively.

Anion exchange membranes have multiple positively charged binding sitesthat attract and hold various impurities and contaminants. The amount ofimpurities that can be potentially removed is a function of theconcentration of these binding sites on the membrane, and the chemicalnature of the ligand (as well as the concentration of these ligands) isresponsible for the strength of binding for the various impurities. Highstrength of binding is a key attribute for increasing the removal ofimpurities, for example, host cell proteins. Strength of binding (SB) isrelated to the ionic strength of solutions required to elute the boundimpurities. SB of membrane sorber (measured in conductivity units,mS/cm) is determined as follows. First, a small amount of adsorbatesolution is passed through the membrane sorber so the adsorbate binds tothe membrane sorber. Second, the membrane sorber is eluted withincreasing gradient of inorganic salt, such as sodium chloride. Theminimum conductivity of elution solution required to elute off theadsorbate is recorded and defined as the SB of that membrane sorber. Byincreasing the sorber strength of binding, negatively charged impuritiescan be made to bind irreversibly to the membrane sorber, therebysignificantly increasing the removal efficiency. This is particularlyimportant for the removal of weakly bound populations of host cellproteins from an antibody stream.

Conventional flow-through anion exchangers typically contain aquaternary ammonium ligand that is responsible for attracting andbinding negatively charged impurities, while repelling the positivelycharged product molecules. Conventional wisdom dictates that in order tobind impurities strongly by charge interaction, a strong anionexchanger, i.e., one that has a positive charge at all pH values, isnecessary. The present invention demonstrates otherwise. The presentinventors found that polymeric primary amines, preferably aliphaticpolymers having a primary amine covalently attached to the polymerbackbone, more preferably having a primary amine covalently attached tothe polymer backbone by at least one aliphatic group, preferably amethylene group, bind negatively charged impurities exceptionallystrongly and thus are the preferred class of materials for creating theadsorptive hydrogel on the surface of a membrane sorber.

Monoclonal antibodies continue to gain importance as therapeutic anddiagnostic agents. The process of screening hybridoma libraries forcandidate mABs is both time consuming and labor intensive. Once ahybridoma cell line expressing a suitable mAB is established, apurification methodology must be developed to produce sufficient mAB forfurther characterization. A traditional method for purifying involvesusing Protein A or Protein G affinity chromatography. The purifiedantibody is desalted and exchanged into a biological buffer usingdialysis. The entire process typically requires several days to completeand can be particularly onerous if multiple mABs are to be evaluated inparallel.

U.S. Pat. No. 6,090,288 teaches preparation of amino group containingchromatography media for separation of peptides and nucleic acids. It isdisclosed that a higher ionic strength is necessary for elution ofproteins from weak anion exchange ligands vs. strong ones. The importantstructural feature of the separation media disclosed is that “at adistance of 2 or 3 atoms away from an amino nitrogen there is a hydroxylgroup or a primary, secondary or tertiary amino group”. Herein, forexample, we show that a loosely crosslinked coating of purepolyallylamine polymer does not require any additional groups to promotehigher strength of binding of proteins.

U.S. Pat. No. 5,304,638 teaches using a protein separation medium thatcomprises a water-insoluble matrix carrying a plurality of polyaminegroupings. One of the examples demonstrates preparing a polyallylaminesurface modified agarose chromatography gel. However, the inventors ofU.S. Pat. No. 5,304,638 fail to recognize the importance of using aprimary amines vs. secondary and tertiary amines. No effort is made ordescribed concerning controlling coating thickness to optimize sorption,or to crosslink the coating for stability. They emphasize the preferrednumber of carbon atoms between pairs of nitrogen atoms as being not morethan 3. They introduce an empirical function Q, which is calculatedbased on the structure of polyamine grouping and pH and has a preferredvalue of at least 1.5. In polyallylamine, there are 5 carbon atomsbetween nearest nitrogen atoms, and the Q function for it would be closeto 0.1.

U.S. Pat. No. 5,547,576 teaches preparing a porous membrane that has animmobilized polyamine coating and could be used to remove viruses fromaqueous solution. The coating preparation involves first grafting aradical on the surface of the membrane and then reacting the radicalwith a polyamine compound. Grafting modifications are often impracticaldue to their inherent complexity: they are sensitive to the particularsubstrate a radical is grafted to, and can be expensive to implement inmanufacturing environment. This method also suffers from the structuraldeficiencies discussed re: 5,304,638

All three of these U.S. Pat. Nos. 6,090,288, 5,304,638, and 5,547,576,fail to recognize the importance of control over the thickness ofpolyamine coating or the degree of polymer cross-linking within thatcoating. All of them rely on chemical reaction of an amine-containingcompound with a reactive group that has been covalently immobilized onthe surface, either by grafting or direct reaction. In the end of anysuch procedure, one is essentially limited to a monolayer-typeamine-containing coating. High sorptive capacity of these membranes canonly be achieved by increasing the surface area, as is the case withagarose chromatography beads. The present invention teaches that highsorptive capacity is achieved by building a relatively thick layer ofloosely cross-linked polymeric layer on the membrane surface, aradically different approach from all those taught in the prior art.

Accordingly, it would be desirable to provide media and a flow-throughanion exchanger including such media, that offer strong binding ofimpurities and that do not suffer from the shortcomings of the priorart. Such an exchanger is particularly useful in the purification ofmonoclonal antibodies from cell culture media using a chromatographyscheme, when placed downstream of an affinity chromatography column thatis optionally followed by one or more polishing steps carried out with acation exchange column, for example.

SUMMARY OF THE INVENTION

The problems of the prior art have been overcome by the presentinvention, which provides media and devices, such as anion exchangersincluding such media, wherein the media is a membrane having a surfacecoated with a polymer such as a polyallylamine. The resulting membranesurprisingly offers stronger binding of protein impurities and superiorremoval of host cell proteins from biological samples than conventionalligands based on quaternary ammonium salts, including trimethylammoniumligands.

Described is a method to significantly increase the sorptive capacity ofmicroporous membranes. Instead of modifying the surface of the membranein a monolayer-like fashion, the entire external and internal poresurfaces are coated with a loosely cross-linked hydrogel. The wet(swollen) thickness of this hydrogel is about 50-100 nm, which issufficient to accommodate several layers of protein molecules. Thus, theadsorptive capacity of a microporous membrane is boosted from about 20mg/ml to 80-100 mg/ml. The type of interactions driving the adsorptionis defined by the specific application in which a given membrane sorberproduct is used. Currently, there is a need for a high-capacity,high-affinity sorber that removes viruses, nucleic acids, endotoxins,and host cell proteins (HCPs) from biological samples such as solutionsof monoclonal antibodies (MABs). A number of chemical moieties bear apositive charge in an aqueous solution, including primary, secondary,and tertiary amines, as well as quaternary ammonium salts. The aminesare positively charged at pH below 11, while the ammonium salts bear thepositive charge at all pH, so these groups are called weak and stronganion exchangers, respectively. The present inventors discovered thatpolymeric primary amines, preferably aliphatic polymers having a primaryamine covalently attached to the polymer backbone, more preferablyhaving a primary amine covalently attached to the polymer backbone by atleast one aliphatic group, preferably a methylene group, bind negativelycharged impurities exceptionally strongly and thus are the preferredclass of materials for creating the adsorptive hydrogel on the surfaceof a membrane sorber.

Also described is a chromatography scheme and method for purifyingmonoclonal antibodies, wherein the anion exchange sorber is placeddownstream of an affinity column (such as Protein A or Protein Gaffinity column) and optionally one or more polishing devices such ascationic exchange columns, or just a cation exchange column. In view ofthe nature of the media in the anion exchange sorber described, littleor no dilution of the cation exchanger pool (or affinity column exchangepool where no cation exchanger is used) is necessary to lower theconductivity of the sample because the present sorber media can operateat higher ionic strengths. The sorber functions well to strongly bindhost cell proteins and other negatively charged impurities in biologicalsamples even at conductivities approaching 15 mS/cm, and at relativelyhigh pH.

As discussed earlier, relatively high device permeability is achievedwithout sacrificing binding capacity because of the convective flow ofthe membrane sorber compared to the diffusive flow of bead-basedsorbers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph of water flux and BSA capacity of 1 mm membranesorber columns prepared from PAH cross-linked with 0.5% PEG-DGE;

FIG. 1B is a graph of water flux and BSA capacity of 1 mm membranesorber columns prepared from with 9 wt. % PAH cross-linked with PEG-DGE;and

FIG. 2 is a graph of BSA capacity of a membrane sorber in the free baseform and the sulfamate ion form in the accelerated shelf life test.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

The present invention relates to a porous chromatographic or sorptivemedia having a porous, polymeric coating formed on a porous,self-supporting substrate, to anionic exchangers including such media,to purification schemes including the sorber, and to methods ofpurification. The media is particularly suited for the robust removal oflow-level impurities from manufactured biotherapeutics, such asmonoclonal antibodies, in manner that integrates well into existingdownstream purification processes. Typical impurities include DNA,endotoxin, HCP and viruses. The media functions well at high saltconcentration and high conductivity (high affinity), effectivelyremoving viruses even under such conditions. High binding capacitywithout sacrificing device permeability is achieved. Indeed, dependingon the coating properties produced by the methods described herein,nucleic acid binding capacities of greater than about 5 mg/ml, orgreater than about 25 mg/ml, or greater than about 35-40 mg/ml, may beachieved. The amount of the anion exchange sorber is much less than thatfor a comparable bead-based process, since large dilutions of the samplebeing loaded on the sorber media are no longer necessary.

The porous substrate has two surfaces associated with the geometric orphysical structure of the substrate. A sheet will have a top and bottomsurface, or a first and a second surface. These are commonly termed“sides.” In use, fluid will flow from one side (surface) through thesubstrate to and through the other side (surface). For hollow fibers andtubular membranes, there is an inside and an outside surface. Flowproceeds from the inside to the outside, or vice versa, depending ondesign and use.

The thickness dimension between the two surfaces is porous. This porousregion has a surface area associated with the pores. In order to preventconfusion related to the terms “surface”, “surfaces”, or “surface area,”or similar usages, the inventors will refer to the geometric surfaces asexternal or facial surfaces or as sides. The surface area associatedwith the pores will be referred to as internal or porous surface area.

Porous material comprises the pores, which are empty space, and thesolid matrix or skeleton which makes up the physical embodiment of thematerial. For example, in a non-woven web, the randomly oriented fibersmake up the matrix and give the web its form. In polymer microporousmembranes, the phase separated polymer provides the matrix. Herein, theinventors discuss coating or covering the surface of the media. Theinventors mean by this that the internal and external surfaces arecoated so as to not completely block the pores, that is, to retain asignificant proportion of the structure for convective flow. Inparticular, for the internal surface area, coating or covering meansthat the matrix is coated or covered, leaving a significant proportionof the pores open.

Absorption refers to taking up of matter by permeation into the body ofan absorptive material. Adsorption refers to movement of molecules froma bulk phase onto the surface of an adsorptive media. Sorption is ageneral term that includes both adsorption and absorption. Similarly, asorptive material or sorption device herein denoted as a sorber, refersto a material or device that both ad- and absorbs.

The porous substrate acts as a supporting skeleton for the adsorptivehydrogel. The substrate should be amenable to handling and manufacturinginto a robust and integral device. The pore structure should provide foruniform flow distribution, high flux, and high surface area. Thesubstrate may be a fiber, a sheet such as a woven fabric, a non-woven, amat, a felt or a membrane. Preferably, the substrate is a sheet formedof a woven or non-woven fabric or a membrane.

Fibers may be of any length, diameter and may be hollow or solid. Theyare not bonded together as a substrate (although they may be formed intoan unitary structure after application of the coating) but areindividual discrete entities. They may be in the form of a continuouslength such as thread or monofilament of indeterminate length or theymay be formed into shorter individual fibers made by chopping fibrousmaterials such as non-woven or woven fabrics, cutting the continuouslength fiber into individual pieces, formed by a crystalline growthmethod and the like.

Non-woven fabrics are flat, porous sheets made directly from separatefibers bonded together by entangling fiber or filaments, thermally orchemically. Typically, nonwoven fabric manufacturers supply media havingfrom 1 to 500 micron mean flow pore (MFP) ratings. For non-wovenfabrics, the porous structure is the entangled fibers, and porosityrefers to the tortuous spaces between and among the fibers. Porosity hasa similar meaning for felted fabrics. A preferred non-woven is byFreudenberg Nonwovens NA of Lowell, Mass. and is type FO2463.

Woven fabrics are produced by the interlacing of warp fibers and weftfibers in a regular pattern or weave style that is at some predefinedangle to each other. Typically the weft is at an angle of about 90degrees to that of the warp. Other commonly used angles include but arenot limited to 30, 45, 60 and 75 degrees. The fabric's integrity ismaintained by the mechanical interlocking of the fibers cause by theweaving process. Drape (the ability of a fabric to conform to a complexsurface), surface smoothness and stability of a fabric are controlledprimarily by the weave style, such as plain, twill, satin, basket weave,leno, etc. In this case, the substrate porosity is the space between thefibers and is of a less tortuous nature.

The substrate also may be formed from a variety of materials includingglass, plastics, ceramics and metals.

Borosilicate glass is one example of a suitable glass. It can be formedas fibers or glass mats.

Various ceramics based on the more conventional silicate chemistries ormore exotic chemistries such as yttrium, zirconia, titanium and the likeand blends thereof can be used. They can be formed into fibers, mats,felts, monoliths or membranes.

Metals such as stainless steel, nickel, copper, iron or other magneticmetals and alloys, palladium, tungsten, platinum, and the like maybemade into various forms including fibers, sintered sheets andstructures, such as sintered stainless steel or nickel filters, wovenscreens and non-woven mats, fabrics and felts such as stainless steelwool.

The preferred substrate is made from synthetic or natural polymericmaterials. Thermoplastics are a useful class of polymers for this use.Thermoplastics include but are not limited to polyolefins such aspolyethylenes, including ultrahigh molecular weight polyethylenes,polypropylenes, sheathed polyethylene/polypropylene fibers, PVDF,polysulfone, polyethersulfones, polyarylsulphones, polyphenylsulfones,polyvinyl chlorides, polyesters such as polyethylene terephthalate,polybutylene terephthalate and the like, polyamides, acrylates such aspolymethylmethacrylate, styrenic polymers and mixtures of the above.Other synthetic materials include celluloses, epoxies, urethanes and thelike.

Suitable substrates include microporous filtration membranes, i.e. thosewith pore sizes from about 0.1 to about 10 μm. Substrate material can behydrophilic or hydrophobic. Examples of hydrophilic substrate materialsinclude, but are not limited to, polysaccharides and polyamides, as wellas surface treated hydrophilic porous membranes, such as Durapore®(Millipore Corporation, Billerica Mass.). Examples of hydrophobicmaterial include, but are not limited to, polyolefins, polyvinylidenefluoride, polytetafluoroethylene, polysulfones, polycarbonates,polyesters, polyacrylates, and polymethacrylates. The porous structureis created from the substrate material by any method known to thoseskilled in the art, such as solution phase inversion,temperature-induced phase separation, air casting, track-etching,stretching, sintering, laser drilling, etc. Because of the universalnature of the present invention, virtually any available method tocreate a porous structure is suitable for making the supporting skeletonfor the membrane sorber. A substrate material made from ultra-highmolecular weight polyethylene has been found to be useful due to itscombination of mechanical properties, chemical, caustic and gammastability.

The coating polymer forms the adsorptive hydrogel and bears the chemicalgroups (binding groups) responsible for attracting and holding theimpurities. Alternatively, the coating polymer possesses chemical groupsthat are easily modifiable to incorporate the binding groups. Thecoating is permeable to biomolecules so that proteins and otherimpurities can be captured into the depth of the coating, increasingadsorptive capacity. The preferred coating polymer is a polymericprimary amine. Examples of suitable polymeric primary amines includepolyallylamine, polyvinylamine, polybutylamine, polylysine, theircopolymers with one another and with other polymers, as well as theirrespective protonated forms. A coating made from polyallylamine (and/orits protonated form, for example polyallylamine hydrochloride (PAH)) hasbeen found to be particularly useful. PAA is commercially available(Nitto Boseki) in a number of molecular weights, usually in the rangefrom 1,000 to 150,000, and all these can be used for creating a membranesorber. PAA and PAH are readily soluble in water. The pH of aqueoussolution of PAA is about 10-12, while that of PAH is 3-5. PAA and PAHmay be used interchangeably, however the pH of the final solution mustbe monitored and if necessary adjusted to the value above 10 so thatnon-protonated amino groups are available for reaction with across-linker.

The coating typically constitutes at least about 3% of the total volumeof the coated substrate, preferably from about 5% to about 10%, of thetotal volume of the substrate. In certain embodiments, the coatingcovers the substrate in a substantially uniform thickness. Suitablethicknesses range of dry coating from about 10 nm to about 50 nm.

A cross-linker reacts with the polymer to make the latter insoluble inwater and thus held on the surface of the supporting skeleton. Suitablecross-linkers are difunctional or polyfunctional molecules that reactwith the coating polymer and are soluble in the chosen solvent, which ispreferably water. A wide variety of chemical moieties react with primaryamines, most notably epoxides, chloro-, bromo-, and iodoalkanes,carboxylic acid anhydrides and halides, aldehydes, α,β-unsaturatedesters, nitriles, amides, and ketones. A preferred cross-linker ispolyethylene glycol diglycidyl ether (PEG-DGE). It is readily soluble inwater, provides fast and efficient cross-linking, and is hydrophilic,neutral, non-toxic and readily available. The amount of cross-linkerused in the coating solution is based on the molar ratio of reactivegroups on the polymer and on the cross-linker. The preferred ratio is inthe range from about 10 to about 1,000, more preferred from about 20 toabout 200, most preferred from about 30 to about 100. More cross-linkerwill hinder the ability of the hydrogel to swell and will thus reducethe sorptive capacity, while less cross-linker may result in incompletecross-linking, i.e. leave some polymer molecules fully soluble.

A surfactant may be used to help spread the polymer solution uniformlyon the entire surface of the supporting structure. Preferred surfactantsare non-ionic, water-soluble, and alkaline stable. Fluorosurfactantspossess a remarkable ability to lower water surface tension. Thesesurfactants are sold under the trade name Zonyl by E.I. du Pont deNemours and Company and are particularly suitable, such as Zonyl FSN andZonyl FSH. Another acceptable class of surfactants are octylphenolethoxylates, sold under the trade name Triton X by The Dow ChemicalCompany. Those skilled in the art will appreciate that other surfactantsalso may be used. The concentration of surfactant used in the coatingsolution is usually the minimum amount needed to lower the solutionsurface tension to avoid dewetting. Dewetting is defined as spontaneousbeading up of liquid on the surface after initial spreading. Dewettingis a highly undesirable event during formation of the membrane sorber,since it leads to non-uniform coating and exposure of the substrate,which sometimes results in non-wettable product and reduced sorptivecapacity. The amount of surfactant needed can be conveniently determinedby measuring contact angles that a drop of solution makes with a flatsurface made from the same material as the porous skeleton. Dynamicadvancing and receding contact angles are especially informative, whichare measured as the liquid is added to or withdrawn from the drop ofsolution, respectively. Dewetting can be avoided if the solution isformulated to have the receding contact angle of 0°.

A small amount of a hydrophilic polymer that readily adsorbs on ahydrophobic surface optionally may be added to the solution as aspreading aid. Polyvinyl alcohol is the preferred polymer and can beused in concentrations ranging from about 0.05 wt. % to about 5 wt. % oftotal solution volume.

When the supporting porous structure cannot be readily wetted with thesolution of polymer, such as in the case of hydrophobic microporousmembrane, a wetting aid can be added to the solution. The wetting aidcan be any organic solvent compatible with the coating polymer solutionthat does not negatively affect the cross-linking reaction. Typicallythe solvent is one of the lower aliphatic alcohols, but acetone,tetrahydrofuran, acetonitrile and other water-miscible solvents can beused as well. The amount of the added organic solvent is the minimumneeded to effect instant wettability of the porous substrate with thecoating solution. Exemplary wetting aids include methyl alcohol, ethylalcohol, and isopropyl alcohol.

Methods of coating can improve the wetting of the web by the coatingsolution. The coating solution may be forced into the web in acontrolled manner so as to uniformly saturate the web and leave nohydrophobic spots or areas. This may be done, for example by extrudingthe solution through a slot pressed against the web, or in closeproximity to the web, in order to force the solution by the appliedpressure of extrusion into the web. Persons skilled in the art will beable to determine conditions of pressure, speed and slot geometry neededto produce a uniform coating.

A preferred process for forming the coated substrate comprises the stepsof: 1) preparing the solution; 2) applying the solution on the membrane;removing excess liquid from the external surfaces of the substrate 3)drying the membrane; 4) curing the membrane; 5) rinsing and drying ofthe membrane; 6) optional annealing of the finished membrane; and 7)optional acid treatment of the membrane. More specifically, a solutionis prepared that contains a suitable polymer and cross-linker. Theconcentrations of these two components determine the thickness anddegree of swelling of the deposited coating, which in turn define fluxthrough the membrane and its sorptive capacity, as shown below. Thepolymer and cross-linker are dissolved in a suitable solvent, preferablywater. The solution may optionally contain other ingredients, such aswetting aids, spreading aids, and a pH adjuster. If a hydrophobicsubstrate is used, the surface tension of solution will have to be lowenough in order to wet it. Aqueous solutions of polymers typically willnot wet hydrophobic microporous membranes, so an organic solvent(wetting aid) will have to be added to the solution. To help spread thecoating uniformly on the surface of a hydrophobic membrane, a surfactantmay be added to the solution. Finally, depending on the chemical natureof the cross-linker, the pH may need to be raised in order to effect thecross-linking reaction. The solution components and typicalconcentration ranges are listed in Table 1:

TABLE 1 Component Role Range, wt. % Polymeric Hydrogel-forming 3-15primary amine adsorptive polymer Cross-linker Effects 0.01-2.0 formation of hydrogel Surfactant Surfactant for  0-3.0 even coatingHydrophilic Surface  0-1.0% polymer hydrophilization Organic Initialwetting 0-30 solvent of hydrophobic membrane with coating solutionInorganic Raise pH to  0-5.0 base effect cross- linking Water SolventBalanceThe coating solution is applied on the substrate such as by submergingthe substrate into solution, removing the substrate from solution, andremoving excess of solution from both sides of the substratemechanically, for example, using a pair of nip rolls (Nipped off). Theporous substrate whose pores are filled with solution is subsequentlydried. Drying can be carried out by evaporation at room temperature orcan be accelerated by applying heat (Temperature range of about 40-110°C.). After the coated substrate is dried, it is held for a period offrom several hours to several days so that cross-linker can fully reactwith the polymer. Cross-linking may be optionally accelerated byapplying heat. The substrate is subsequently rinsed with copious amountsof solvent and dried again. Additional optional process steps includeannealing the dried membrane sorber at an elevated temperature (60-120°C.) to adjust its flow properties and treating it with a strongnon-oxidizing monobasic acid at concentration 0.1M to 1M to protonatethe amino groups present in the coating.

Where the polymer is PAA, converting essentially all amino groups in thepolymer into corresponding ammonium salts after heat treatment of themembrane will help ensure consistency of the product. Good waterwettability is important. Since the base material is very hydrophobicand the hydrophilic coating is very loosely cross-linked and notcovalently attached to the matrix, some lateral shrinking of the PAA gelwill cause the membrane to become not wettable with water. On the otherhand, if essentially all amino groups of the PAA are converted intocorresponding ammonium salts, the increased volume of the dried coating,greater retention of water by counter-ions and stronger affinity forwater of the charged polymer will help to make the membrane morewater-wettable. A strong, non-toxic, non-oxidizing acid, preferably onethat is monobasic to avoid ionic cross-linking of PAA, should be used toprotonate PAA for this purpose. Suitable acids include hydrochloric,hydrobromic, sulfamic, methansulfonic, trichloroacetic, andtrifluoroacetic acid. Although chloride may be the counter-ion of choicesince it is already present in the sample protein solution, it may notbe practical for a continuous process to use hydrochloric acid and/orits salt due to the corrosion of steel and the occupational safetyissues involved. A more suitable acid is thus sulfamic acid (H₂N—SO₂OH)is preferred as the protonating agent for PAA.

A suitable process for protonating the PAA is to submerge the membranein a 0.1-0.5 M solution of the protonating acid, preferably sulfamicacid in water (or a water/alcohol mix to fully penetrate a poorlywetting membrane), followed by rinsing and drying. The resultingmembrane will bear sulfamate counter-ions, which may be easily exchangedout by employing a simple conditioning protocol, such as 0.5M sodiumhydroxide followed by 0.5M sodium chloride.

Such acid treatment improves shelf life stability of the membrane, andalso results in a significantly higher strength of binding. Although thepresent inventors should not be limited to any particular theory, it isbelieved that when PAA is dried in the fully protonated (acid-treated)state, it assumes a more extended, “open” morphology that is capable ofbetter encapsulating BSA and thus will not release it until a higherionic strength is reached. A further benefit of acid-treated membranesis greater stability towards ionizing irradiation, such as gammairradiation, which is an accepted sterilization procedure for filtrationproducts.

Three critical parameters define a successful membrane sorber product.They are: sorptive capacity, flux, and strength of binding. While thestrength of binding is to a large extent determined by the chemicalnature of groups presented on the surface of membrane sorber, capacityand flux are usually a lot more sensitive to the procedure employed toform the sorptive layer and the amount of polymer and cross-linker. Itis often observed that for a given combination of supporting skeleton,purification efficiency (determined by the bed height) and chemicalnature of the membrane sorber, greater flux can translate into smallersorptive capacity, and vice versa.

FIGS. 1A and 1B show typical trends observed for a membrane sorberprepared according to the present invention. From these graphs, thetrade-off between flux and capacity is obvious. It is seen that both PAHand cross-linker have a direct impact on these critical properties ofthe membrane sorber. To perform satisfactorily in the application, agood membrane sorber should possess both high flux and high capacity.Flux of a membrane sorber can be generally expressed in chromatographicunits, CV/min/psi, where CV is Column Volume. The flux of a given columnvolume obviously depends on the column height, which is usuallyoptimized for efficient separation. In case of an anion-exchangemembrane sorber, efficient column height can be defined by the minimumheight required to effect 99.99% removal (LRV 4.0) of a negativelycharged virus, for example MMV. This column height was found to be about0.5 mm for the membrane sorber in the present invention. For addedassurance of virus clearance, columns of double that height (1 mm) areused routinely throughout this invention. A desirable flux of a membranesorber with such column height would be at least 2.0 CV/min/psi orbetter, more preferably 2.5 CV/min/psi or better. Achieving the rightcombination of flux and capacity is exceptionally difficult. The presentinventors have had to go significantly beyond routine experimentation orconcentration optimization to achieve the outstanding properties of themembrane sorber. For example, the cross-linker was judiciously chosen tobe a highly flexible, polymeric molecule that proven very beneficial forhigh sorptive capacity. Using intermediate molecular weight of PAA(15,000) has allowed achieving a balance between flow and capacity. Theinorganic base used to adjust pH was of non-salting-out kind so that ahigher concentration of PAA could be used to achieve high capacity. Thechoice of surfactant was dictated by recognizing the need for anon-ionic, caustic-stable compound, while the amount of surfactantneeded was found in a separate investigation. For contrast, a PAAmembrane sorber prepared in accordance with pending application US2005/0211615 has flux of just 0.5 CV/min/psi for 1 mm column, while theBSA capacity is only about 61 mg/ml.

Another important aspect of this invention is the post-treatmentprocedure employed after the sorber membrane is cured, rinsed, anddried. The inventors discovered that treatment of membrane sorber basedon polymeric primary amines with acid significantly boosts its strengthof binding, wettability, and stability towards ionizing radiation.

A very surprising discovery made by the inventors was that acidtreatment significantly increases the strength of binding of membranesorber, from about 54-58 mS/cm to 78-82 mS/cm. It could be hypothesizedthat when PAA is dried in the fully protonated (acid-treated) state, itassumes a more extended, “open” morphology that is capable of betterencapsulating BSA and thus will not release it until a higher ionicstrength. This benefit was unintended and surprising, yet it is verybeneficial since a higher SB usually translates into better removal oftrace impurities.

The permeability of the cross-linked PAA membrane adsorber was improvedby a high-temperature “curing” process. The lightly cross-linked PAA-gelhas the ability to absorb significant amount water resulting in ordersof magnitude increase in its volume. This effect can cause lowpermeability. It appears that this property of the gel is reduced bydehydrating it to such an extent that it reduces the swelling to anacceptable level, without compromising the strength of binding andcapacity of the gel. In fact, the curing process is capable of tuningthe permeability of the membrane as necessary for the product. Suitablecuring temperatures are about 25-120° C., more preferably from about85-100° C.; and for about 6 to 72 hours.

Gamma irradiation is a widely accepted sterilization procedure forfiltration products. Gamma-sterilizability is a desirable feature of amembrane sorber. The inventors observed a surprising benefit ofacid-treated membrane sorber in the fact that it had a greater stabilitytowards ionizing irradiation. FIG. 2 demonstrates BSA capacity ofmembrane sorber samples (control, not treated with acid) and thoseconverted into ammonium sulfamates. All of these samples were irradiatedwith 25 kGy of electron beam to simulate gamma sterilization conditions.It is clear that acid-treated samples maintain their properties muchbetter than the non-acid treated.

The thus formed anion exchange sorber is particularly useful when placedin a Mab purification chromatography scheme. For example, cell culturefluid including monoclonal antibodies can be purified using affinitychromatography, such as a Protein A or Protein G affinitychromatography, followed by one or more cation exchange columns. Theoutput from the final cation exchange column then can be flowed throughthe present anion exchange column for significantly reducing theconcentration of residual host cell proteins, viruses, nucleic acids,endotoxins and other impurities in the fluid by causing these impuritiesto bind to the media under appropriate conditions while allowing useful,purified product to flow through the exchanger.

Importantly, further purification of the cation exchange pool (theoutput from the cation exchange column(s)) using the instant anionexchange sorber can be accomplished without the significant dilution ofthe cation exchange pool that was conventionally necessary in order toreduce the salt concentration (and lower conductivity) to enable thehost cell proteins to effectively bind. Indeed, the sorbers of thepresent invention have very high binding capacity even at high saltconcentrations and conductivity (e.g., >60 g/L BSA binding at 200 mMNaCl), and allow for much higher mass loading than conventional sorbers(e.g., 3 kg/L compared to 0.05 kg/L) under typical cation exchange poolconditions, see Example 7 below. The ability to reduce or eliminatedilution of the cation exchange pool due to the high capacity at highsalt concentrations of the instant sorber is a significant advantage.

The present sorber also exhibits robust removal of viruses at relativelyhigh salt concentration and relatively high pH (pH's of 7.6 and saltconcentrations of 100 mM and 150 mM still resulted in acceptable removalof virus).

Those skilled in the art will also appreciate that for someapplications, the downstream polishing carried out by the one or morecation exchange columns may not be necessary, and the instant anionexchange sorber can be placed downstream of the affinity column withoutintermediate cation exchange columns in between. Analogously, for someapplications the instant anion exchange sorber can be placed downstreamof the cation exchange column with no need for capture (affinity) columnpreceding it.

The following examples are included herein for the purpose ofillustration and are not intended to limit the invention.

Example 1 PAA on Hydrophobic UPE

An aqueous solution was prepared containing 20 wt. % isopropanol, 9 wt.% PAH (molecular weight 15,000), 3 wt. % lithium hydroxide monohydrate,2 wt. % Triton X-100, 0.5 wt. % PEG-DGE (molecular weight 526), and 0.2wt. % polyvinyl alcohol (molecular weight 30,000, degree of hydrolysis98%). A hydrophobic UPE membrane with pore size rating 0.65 μm was fullywetted with this solution, and excess liquid was nipped off. Themembrane was dried at room temperature for 24 hours and rinsed withcopious amounts of water and methanol and dried again. The membrane wetsreadily with water and has weight add-on of about 25%. An eight-layerMillex®-type syringe device was manufactured from this membrane that hasa surface area of 3.5 cm² and column height 1 mm. The device has a flowrate of 4.3 CV/min/psi, BSA capacity 90 mg/ml, and strength of bindingof 54 mS/cm.

Comparative Example 1 PAA-GTMAC on Hydrophobic UPE

The modified membrane from Example 1 was further modified by submergingin 50 wt. % of glycidyltrimethylammonium chloride (GTMAC) at pH 13 for24 hours, rinsed with water and dried. An eight-layer Millex®-typesyringe device was manufactured from this membrane that has a surfacearea of 3.5 cm². The device has a flow rate of 0.7 CV/min/psi, BSAcapacity 80 mg/ml, and strength of binding of 19 mS/cm.

Comparative Example 2 Sulfamic Acid Treatment

The modified membrane from Example 1 was further modified by submergingin 0.5M solution of sulfamic acid in water for 10 minutes, rinsed withdeionized water and dried. An eight-layer Millex®-type syringe devicewas manufactured from this membrane that has a surface area of 3.5 cm².The device has a flow rate of 4.3 CV/min/psi, BSA capacity 80 mg/ml, andstrength of binding of 80 mS/cm.

Comparative Example 3

An aqueous solution was prepared containing 11.6 wt. % PAH (molecularweight 75,000), 18.6 wt. % 1.0N sodium hydroxide solution, 11.6% sodiumchloride, 23.2% of 17% aqueous solution of epichlorohydrin-modifiedpolyethylenemine. A hydrophobic UPE membrane with pore size rating 0.65μm was prewet with methanol and directly contacted with the aboutsolution for 5 minutes. The wet membrane was placed in a polyethylenefilm bag and the bag was placed in an oven at 85° C. for 7 minutes whilebeing careful not to dry out the membrane to initiate the crosslinkingreaction. The wet membrane was then removed from the bag and allowed todry at room temperature. The dry membrane was then placed in an oven at100° C. for four hours to complete the crosslinking reaction. Themembrane was then thoroughly washed with water, methanol andhydrochloric acid (1.0 N) and allowed to dry at room temperature. Themembrane did not wet with water. An eight-layer Millex®-type syringedevice was manufactured from this membrane that has a surface area of3.5 cm². The device has a flow rate of 0.5 CV/min/psi, BSA capacity 61mg/ml, and strength of binding of 81 mS/cm.

Example 2 PAA on Hydrophilic UPE

An aqueous solution was prepared containing 10 wt. % PAA (molecularweight 15,000) and 0.5 wt. % PEG-DGE (molecular weight 526). Ahydrophilic UPE membrane with pore size rating 0.65 μm was fully wettedwith this solution, and excess liquid was nipped off. The membrane wasdried at room temperature for 24 hours, rinsed with copious amounts ofwater and dried again. The eight-layer device has a flow rate of 4.3CV/min/psi, BSA capacity 80 mg/ml, and strength of binding of 49 mS/cm.

Example 3 Polyvinyl Amine on Hydrophilic UPE

An aqueous solution was prepared containing 20 wt. % isopropanol, 7 wt.% polyvinyl amine (Lupamin 9095, BASF Corp., Mount Olive, N.J.) and 0.5wt. % PEG-DGE (molecular weight 526). A hydrophilic UPE membrane withpore size rating 0.65 μm was fully wetted with this solution, and excessliquid was nipped off. The membrane was dried at room temperature for 24hours and rinsed with copious amounts of water and methanol and driedagain. An eight-layer device was manufactured from this membrane thathas a surface area of 3.5 cm². The device has a flow rate of 3.1CV/min/psi, BSA capacity 87 mg/ml, and strength of binding of 32 mS/cm.

Example 4 Polylysine on Hydrophobic UPE

An aqueous solution was prepared containing 20 wt. % isopropanol, 4 wt.% polylysine hydrobromide (molecular weight 30,000-50,000), 4 wt. %lithium hydroxide monohydrate, 1 wt. % Triton X-100, 0.25 wt. % PEG-DGE(molecular weight 526), and 0.1 wt. % polyvinyl alcohol (molecularweight 30,000, degree of hydrolysis 98%). A hydrophobic UPE membranewith pore size rating 0.65 μm was fully wetted with this solution, andexcess liquid was nipped off. The membrane was dried at room temperaturefor 24 hours and rinsed with copious amounts of water and methanol anddried again. The membrane did not wet with water and had weight add-onabout 9%. An eight-layer device was manufactured from this membrane thathas a surface area of 3.5 cm². The device had a flow rate of 7.1CV/min/psi, BSA capacity 34 mg/ml, and strength of binding of 32 mS/cm.

Example 5 PAA on Activated Sepharose Beads

Agarose chromatography beads were modified with PAA. 2 g of EpoxySepharose 6B (GE Healthcare) were suspended in 8 ml Milli-Q water, towhich was added 10 ml of 10% solution of PAA (molecular weight 3,000).The pH was adjusted to 11 with sodium hydroxide. The beads were gentlyshaken for 24 hours, carefully rinsed with water and stored wet inrefrigerator prior to use. A chromatography column was packed withmodified beads (column height 5.5 cm, column volume 1.88 mL) and tested.It had BSA capacity 20.6 mg/ml and SB 83 mS/cm.

Comparative Example 4 PAA-GTMAC on Activated Sepharose Beads

PAA-modified agarose beads from Example 5 were further modified byreacting them with 2% solution of GTMAC at pH 11 overnight. The beadswere carefully rinsed with water and stored wet in refrigerator prior touse. A chromatography column was packed with modified beads (columnheight 4.7 cm, column volume 1.61 mL) and tested. It had BSA capacity87.3 mg/ml and SB 25.4 mS/cm.

Example 6

Membranes with immobilized PAA from Example 1 were sealed in 8-layer,syringe devices and tested for virus retention. The data in Table 2below show the impact of increasing salt concentration on MMV and MuLVretention. As seen in the Table, the MMV data for the Pall Mustangmembrane (strength of binding 20 mS/cm) show that the LRV drops withincreasing conductivity, whereas for the membrane of the invention(i.e., high strength of binding), complete removal is observed, even atthe elevated salt concentrations.

In addition, the HCP removal was significantly better for the PAAmembranes as compared to the quaternary ammonium salt chemistry(PAA-GTMAC, manufactured according to Comparative Example 1), as seen inTable 3. For the data in Table 3, the control was Capto Q beads sold byGE Amersham, which has a medium strength of bonding (i.e., approximately30 mS/cm). It should be noted that these LRV's were obtained withoutsignificant losses of product due to non-specific binding of themonoclonal antibodies to the functionalized membrane (i.e., theanion-exchange properties effectively repelled the product from themembrane surface).

TABLE 2A Virus removal (X-MuLV) as a function of Salt ConcentrationX-MuLV LRC DEVICE 50 mM Salt 100 mM Salt PAA >3.9 >3.7 Pall >3.9 >3.7

TABLE 2B Virus removal (MMV) as a function of Salt Concentration and pHNaCl Concentration in 25 mM Tris buffer 50 100 150 pH 7.6 pH 7.1 pH 7.6pH 7.1 pH 7.6 pH 8.1 (7.2 (12.2 (12.1 (17.2 (17.0 (16.6 Device mS/cm)mS/cm) mS/cm) mS/cm) mS/cm) mS/cm) PAA >6.2 >5.3 >5.3 >5.5 >5.5 >5.4membrane sorber Pall >6.2 2.1 2.8 0.6 0.9 1.8 Mustang Q

TABLE 3 HCP removal from three different Monoclonal Antibodies PrototypeCon- (PAA from duc- HCP Example Quaternary tivity Load 1) Capto Qammonium Product (mS/ (ng/ Removal Removal Removal (Pro. A Pool) cm) ml)(@ 400 CV) (@ 30 CV) (@ 400 CV)   4 g/L Mab 1 10.7 420 62% 14% 0%   7g/L Mab 2 9.7 1800 31% 0% 0% 3.7 g/L Mab 3 22 200 85% 30% 0%

Example 7

A 0.65 μm UPE membrane was coated with 9% PAA and 0.5% PEGDGE on thepilot coating machine. The membrane was extracted and dried at 85° C. ina convection oven for 10 minutes. One sample was left at 85° C. for 12hrs and the other was stored at room temperature. The membranes werethen treated with 5% sulfamic acid in 5% isopropyl alcohol to stabilizethe coating. The permeability of the membranes was then measured using aflux tester. In this test, the time taken for 500 ml of water to passthrough a membrane sample (cross sectional area 9.6 sq.cm.) under vacuum(27.0″ Hg) is measured. The membrane lots listed in Table 1 weremanufactured to demonstrate the improvement in permeability as a resultof the curing step. As seen in Table 4, the “non-cured” membranes havesignificantly higher flow-time (indicating lower permeability) than the“cured” membranes.

As seen from Table 5, the curing step can be used to control thepermeability and capacity of the membranes as desired. All the membraneshad a BSA strength of binding between 60-75 mS.

TABLE 4 Comparison of flow times for cured and non-cured membranes LotNo Curing conditions Flux (s/500 ml) 020307-60821 — 1180 020307-60821 85C. 12 hr 96 020307-60803 — 2650 020307-60803 85 C. 12 hr 106

TABLE 5 Processing Conditions for Membrane Adsorber Cure Cure Flow UPEPAA Roll temp Time Time Lot No Lot No. No (° C.) (hr) (s/500 ml) UPDP60821    103A 100 16 58 101006R9 UPDP 60821   103B 90 6 95 101006R9 UPDP60803 104 90 6 102 101006R6 UPDP 60821 105 90 6 90 101106R5 UPDP 60801102 90 6 84 101106R5 UPDP 60821 011507-1 90 12 80 101006 UPDP 60821011507-2 95 12 52 101106R5

Example 8

0.65 μm UPE membrane rolls were coated with 9% PAA and 0.35% PEGDGE onthe pilot coating machine. The membranes were extracted with water andmethanol and dried on a hot air impingement dryer at 120° C. The rollswere then subjected to curing at varying temperatures and times as shownin the table below. The membranes were then treated with 5% sulfamicacid in 5% isopropyl alcohol to stabilize the coating. The permeabilityof the membranes was measured using a flux tester as described in theprevious example.

As seen in the table below, flow-times on the order of 50 s can beobtained by heating the membrane to temperatures greater than 90° C. for12 hours or longer. In case membranes with lower permeability (higherflow times on the order of 100 s) are desired, the temperature can belimited to below 85° C. and for shorter times (<10 h).

Cap, Flowtime (mg/ml, s) 8 hrs 10 hrs 12 hrs 20 hrs 24 hrs 85° C. 107.7,147.6   95, 63.9 92.9, 88.0 90° C. 95, 41 90, 39 95° C. 92.6, 79.2 89.6,50.2 85.8, 67.1 100° C.  79, 39 63, 41

1. A method of manufacturing an adsorber membrane, comprising providinga substrate having a first external side and a second external side,both sides being porous, and a porous thickness between them;substantially covering said substrate and said first and second externalsurfaces with a sorptive material comprising a crosslinked polymerhaving attached primary amine groups; and heating the resultingsubstrate to a temperature of from about 25° C. to about 120° C.
 2. Themethod of claim 1, wherein said temperature is from about 85° C. toabout 100° C.
 3. The method of claim 1, further comprising treating theresulting membrane with acid, and subjecting the acid-treated membraneto gamma irradiation.
 4. The method of claim 3, wherein said acid isselected from the group consisting of hydrochloric, hydrobromic,sulfamic, methansulfonic, trichloroacetic, and trifluoroacetic acid. 5.The method of claim 3, wherein said acid is sulfamic acid.